title: In vitro evolution of α-hemolysin using a liposome display.
abstract: In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g., antibodies, enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method called liposome display that can evolve the properties of membrane proteins entirely in vitro. This method, which involves in vitro protein synthesis inside liposomes, which are cell-sized phospholipid vesicles, was applied to the pore-forming activity of α-hemolysin, a membrane protein derived from Staphylococcus aureus. The obtained α-hemolysin mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the WT. Given the ability to synthesize various membrane proteins and modify protein synthesis and functional screening conditions, this method will allow for the rapid and efficient evolution of a wide range of membrane proteins.
authors.affiliation: Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujii
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sunami
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kazuta
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D001427:Bacterial Toxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008081:Liposomes / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D017354:Point Mutation; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008081:Liposomes; D010743:Phospholipids; C021091:staphylococcal alpha-toxin
keywords: FACS; Giant unilamellar vesicles; PURE system; directed evolution; in vitro synthetic biology